I have a lot of difficulties measuring succinate-CoA ligase (with ATP/ADP or GTP/GDP) in fibroblast homogenate or in mitochondria isolated from fibroblasts. I tested all the published methods (at 235 nm or using coupling reaction with lactate dehydrogenase and pyruvate kinase) but none were successful (no detectable activity). I tried also to follow the appearance of coenzyme A with Ellman’s reagent by the forward reaction but it seems that succinyl-CoA is unstable and self-hydrolyzes quickly. Can someone help me?
I do not think that succinyl-CoA will be so unstable for self-hydrolysis. Is the enzyme there? Succinyl-CoA ligase can catalyzed the reaction in opposite direction, as a hydrolase...
Are you adding succinate and CoA along with ATP/GTP to the lysate? Since those reagents are critical I would order fresh reagents and try again. I you give it high enough concentrations of substrates then you should force it to make succinate-CoA "until" the succinate-CoA concentration rises to a level that will drive the reaction in the opposite direction to make free succinate and CoA. So initially a time course would be required for the assay. Also, are you using any protease inhibitors in the lysates? I agree with Francisco in that succinate-CoA should not self-hydrolyze, but as he pointed out the the enzyme can catalyze the forward and reverse reactions.
salut Dominique!! A agree with Gary, probably the 2 reactions are occurring and that would prevent to see any changes... Have you add Mg2+ as a cofactor? The protease inhibitors usually help a lot... Best!
Do you have a lot of thiol (e.g. dithiothreitol) in your assay. I have seen instability of acetyl-CoA in the presence of 10 mM dithiothreitol.
Could you please give the concentrations of the components in the coupled assay (with lactate dehydrogenase and pyruvate kinase)? High concentrations of ATP or GTP migh inhibit your enzyme.
Maybe the following publication might help.
Article Novel Reaction of Succinyl Coenzyme A (Succinyl-CoA) Synthet...
To my experience, succinyl-CoA is quite unstable to self-hydrolysis at a pH higher than 5. Thus you should measure the reaction towards succinyl-CoA formation.
As it is longer ago that you asked your question: Did you solve your problem?
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